Treatment of diseases associated with hepatic stellate cell activation using ammonia-lowering therapies

ABSTRACT

Disclosed herein are methods of preventing, treating, and delaying the onset or progression of diseases associated with hepatic stellate cells (HSCs), such as non-alcoholic fatty liver disease (NAFLD), fibrosis, and liver cancer, using ammonia-lowering therapies.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/026,403, filed Jul. 3, 2018, to be issued as U.S. Pat. No.10,525,029, which is a continuation of U.S. patent application Ser. No.15/527,999, filed on May 18, 2017, now U.S. Pat. No. 10,039,735, whichis a U.S. national phase under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2015/062223, filed on Nov. 23, 2015, which claimsthe benefit of priority to U.S. Provisional Application No. 62/083,814,filed on Nov. 24, 2014, each of which is herein incorporated byreference in its entirety.

BACKGROUND Field

The present application relates to the fields of pharmaceuticalchemistry, biochemistry and medicine. One aspect relates to thetreatment and/or prevention of diseases associated with hepatic stellatecell (HSC) activation using ammonia-lowering therapies.

Description of the Related Art

Hepatic stellate cells (HSCs) are pericytes found in the perisinusoidalspace of the liver. Within the liver, stellate cells play an importantrole in maintaining architectural integrity of the liver and areinvolved in fibrosis and liver cancer development. In normal liver, HSCsare in a quiescent state. When the liver is damaged, HSCs can changeinto an activated state. The activated stellate cell is characterized byproliferation, contractility and chemotaxis. Various diseases can resultfrom the activation of HSCs, for example, non-alcoholic fatty liverdisease (NAFLD), fibrotic conditions, and liver cancer.

Various prevention, treatment and management strategies for diseasesassociated with the activation of HSCs are currently available dependingupon the severity of the symptoms. There is a need for additionaltherapies for treating or preventing those diseases.

SUMMARY

Some embodiments disclosed herein provides a method of treating adisease associated with hepatic stellate cell (HSC) activation, whereinthe method comprises performing an ammonia-lowering therapy on a subjectin need thereof. Also disclosed is a method of delaying the onset orprogression of a disease associated with HSC activation, wherein themethod comprises performing an ammonia-lowering therapy on a subject inneed thereof. In some embodiments, performing the ammonia-loweringtherapy comprises administering an ammonia-lowering agent to thesubject.

In some embodiments, the disease associated with HSC activation isnon-alcoholic fatty liver disease (NAFLD). The NAFLD can be, forexample, non-alcoholic steatohepatitis (NASH) or steatosis.

In some embodiments, the disease associated with HSC activation is livercancer. In some embodiments, the disease associated with HSC activationis a fibrotic condition. The fibrotic condition can be, for example,liver fibrosis. In some embodiments, the subject is suffering fromnon-alcoholic fatty liver disease (NAFLD).

Some embodiments provide a method of preventing non-alcoholic fattyliver disease (NAFLD), wherein the method comprises performing anammonia-lowering therapy on a subject in need thereof. For example, theNAFLD can be non-alcoholic steatohepatitis (NASH) or steatosis. In someembodiments, performing the ammonia-lowering therapy comprisesadministering an ammonia-lowering agent to the subject.

In some embodiments, the ammonia-lowering agent is, or comprises, amagnesium phosphate product (MGP), glycerol phenylbutyrate (GPB), sodiumphenylacetate, sodium phenylbutyrate (NaPBA), glutamine, sodiumbenzoate, L-arabinose, a laxative, an antibiotic, ornithine incombination with at least one of phenylacetate and phenylbutyrate, orany combination thereof. In some embodiments, the ammonia-lowering agentis, or comprises, ornithine in combination with at least one ofphenylacetate and phenylbutyrate.

In the methods disclose herein, in some embodiments, separatepharmaceutically acceptable salts of the ornithine and at least one ofphenylacetate and phenylbutyrate are administered to the subject. Insome embodiments, at least one of phenylacetate and phenylbutyrate isadministered as a sodium phenylacetate or sodium phenylbutyrate. In someembodiments, the ornithine is administered as a free monomeric aminoacid or physiologically acceptable salt thereof. In some embodiments,the ornithine and phenylacetate is administered as ornithinephenylacetate.

In some embodiments, the administration is oral, intravenous,intraperitoneal, intragastric, or intravascular administration. In someembodiments, the administration is intravenous administration. In someembodiments, the administration is oral administration

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show ammonia reduces in a dose dependent manner cellproliferation and metabolism in primary human Hepatic Stellate Cells invitro. FIG. 1A shows ammonia inhibits DNA synthesis (BrdU) and metabolicactivity (MTS), and FIG. 1B shows the inhibition is achieved withoutinducing cell death. FIG. 1C shows that ammonia induced strongmorphological changes in a dose-dependent manner i.e. frommyofibroblast-like cells into spindle like fibroblasts as was observedby light microscopy and by Neutral Red cell viability test (20×, 40×).Bar graphs show means of three independent values±SD. *P<0.05, **P<0.01and ***P<0.001 vs. corresponding values of serum free medium (SFM).

FIGS. 2A-F show ammonia induces alterations in cytoplasmic stress, whichcoincides with changes in cellular metabolism/function, contraction, andactin cytoskeleton architecture. FIG. 2A are Transmission ElectronMicroscopy (TEM) images showing that ammonia in a dose-dependent mannercaused dramatic morphological changes with appearance of cytoplasmicvacuoles (V=vacuoles; N=nucleus). FIG. 2B shows recovery of cellproliferation after depletion of ammonia-rich culture medium. Bar graphsshow means of three independent values±SD. *P<0.05 and ***P<0.001 vs.SFM.

FIGS. 2C and 2D depicts results from a collagen gel contraction assayshowing that ammonia induces hHSC contraction. Bar graphs show means of2 independent experiments (values±SD. *P<0.05 and **P<0.01 vs.corresponding values of SFM. FIG. 2E shows that ammonia-induced HSCcontraction coincides with changes in morphology. FIG. 2F shows thatprolonged treatment (72 h) with ammonia induces in a dose-dependentmanner the re-organization of filamentous actin (TRITC-Phalloidinstaining).

FIGS. 3A-C show ammonia induces ROS production. FIG. 3A shows thatprolonged treatment of hHSC with ammonia for 72 hours induces ROSproduction in hHSC. The formation of reactive oxygen species (ROS) wasmeasured using Image-IT™ LIVE Green Reactive Oxygen Species DetectionKit. In FIG. 3B, mean fluorescence intensity (MFI) of ROS signal wasnormalized according to the number of cells (Hoechst 33342), andexpressed as percentage of control. Bar graphs show means of threeindependent values±SD. ***P<0.001 vs corresponding values of SFM. FIG.3C shows that jyperammonemia increases mRNA expression level of ROSmarker SOD2 at 3 and 24 hours. Bar graphs show means of 2 independentvalues±SD. ***P<0.001 vs corresponding values of SFM. FIG. 3D showsNAC-induced ROS scavenger reduces ammonia-induced SOD2 mRNA expressionat 24 hours. Bar graphs show means of 2 independent values±SD.*P<0.05**P<0.01 vs corresponding values of SFM.

FIGS. 4A-C show ammonia modifies mRNA expression and protein level ofseveral pro-inflammatory and HSC activation markers. FIG. 4A shows thatammonia affects protein expression of α-SMA, vimentin, PDGF-Rβ, MyosinIIa and IIb, and p-38 MAPK. FIG. 4B shows that ammonia inducesup-regulation of MMP2 mRNA whereas TIMP1 mRNA is down-regulated. FIG. 4Cshows that Interleukin 1β and Interleukin IL6 mRNA expression areupregulated. Bar graphs show means of three independent values±SD.*P<0.05, **P<0.01 and ***P<0.001 vs. corresponding values of SFM.

FIGS. 5A-B show hyperammonemia treatment further enhances BDL-inducedHSC markers in vivo. FIG. 5A shows that plasma levels of ammonia aresignificant upregulated in BDL and AAs-fed BDL animals in comparison tosham operated rats (*P<0.05 and **P<0.01 vs Sham). OP treatment reducessignificant ammonia in BDL-AAs-fed animals in comparison to BDL animals(**P<0.01). FIG. 5B shows that hyperammonemia treatment in BDL-inducedfibrosis showed an additional significant increase in Myosin IIb,Collagen type I and PDGF-Rβ protein expression in comparison toBDL-induced fibrosis (**P<0.01 and ***P<0.001). In contrast, treatmentwith OP, abrogated the strong effect of AAs-fed BDL on all HSC-relatedactivation markers (*P<0.05, **P<0.01 and ***P<0.001).

FIG. 6 shows expression of ornithine transcarbamylase (OTC) protein andgene in rats fed a normal and a high-fat high-cholesterol diet for 10months and recovery for 2 months.

FIG. 7 shows OTC gene expression in mice fed a methionine-cholinedeficient diet (MCD) for 4 weeks and treated with or without carbon.

FIG. 8 shows OTC gene expression in NAFLD human patients with simplesteatosis or NASH+fibrosis during bariatric surgery.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. The illustrativeembodiments described in the detailed description, drawings, and claimsare not meant to be limiting. Other embodiments may be utilized, andother changes may be made, without departing from the spirit or scope ofthe subject matter presented here. It will be readily understood thatthe aspects of the present disclosure, as generally described herein,can be arranged, substituted, combined, and designed in a wide varietyof different configurations, all of which are explicitly contemplatedand make part of this disclosure.

Definitions

As used herein, a “subject” refers to an animal that is the object oftreatment, observation or experiment. “Animal” includes cold- andwarm-blooded vertebrates and invertebrates such as fish, shellfish,reptiles and, in particular, mammals. “Mammal” includes, withoutlimitation, mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats;cows; horses; primates, such as monkeys, chimpanzees, and apes, and, inparticular, humans.

As used herein, a “patient” refers to a subject that is being treated bya medical professional, such as a Medical Doctor (i.e. Doctor ofAllopathic medicine or Doctor of Osteopathic medicine) or a Doctor ofVeterinary Medicine, to attempt to cure, or at least ameliorate theeffects of, a particular disease or disorder or to prevent the diseaseor disorder from occurring in the first place.

As used herein, “administration” or “administering” refers to a methodof giving a dosage of a pharmaceutically active ingredient to avertebrate.

As used herein, a “unit dosage” refers to an amount of therapeutic agentadministered to a patient in a single dose.

As used herein, a “daily dosage” refers to the total amount oftherapeutic agent administered to a patient in a day.

As used herein, “therapeutically effective amount” or “pharmaceuticallyeffective amount” is meant an amount of therapeutic agent, which has atherapeutic effect. The dosages of a pharmaceutically active ingredientwhich are useful in treatment are therapeutically effective amounts.Thus, as used herein, a therapeutically effective amount means thoseamounts of therapeutic agent which produce the desired therapeuticeffect as judged by clinical trial results and/or model animal studies.

As used herein, a “therapeutic effect” relieves, to some extent, one ormore of the symptoms of a disease or disorder. For example, atherapeutic effect may be observed by a reduction of the subjectivediscomfort that is communicated by a subject (e.g., reduced discomfortnoted in self-administered patient questionnaire).

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a compound or pharmaceutical composition to a subject forprophylactic and/or therapeutic purposes. The term “prophylactictreatment” refers to treating a subject who does not yet exhibitsymptoms of a disease or condition, but who is susceptible to, orotherwise at risk of, a particular disease or condition, whereby thetreatment reduces the likelihood that the patient will develop thedisease or condition. The term “therapeutic treatment” refers toadministering treatment to a subject already suffering from a disease orcondition.

Abbreviations

BDL=bile duct ligation.

OP=ornithine, phenylacetate

OTC=ornithine transcarbamylase

GS=glutamine synthetase

HSC=hepatic stellate cell

Ammonia-Lowering Therapies

Disclosed herein are various ammonia-lowering therapies that can be usedto reduce the ammonia level in a subject. For example, one or moreammonia-lowering agents can be used in the therapy to reduce the ammonialevel in the subject. As used herein, the term “ammonia-lowering agent”refers to a substance that can be used to lower the ammonia level in asubject. The mechanism by which the ammonia-lowering agent lowers theammonia level can vary. For example, the ammonia-lowering agent maylower the ammonia level in a subject by reducing the generation ofammonia in the subject, or by absorbing the ammonia in the subject, ordrawing ammonia into the colon and removing ammonia through a laxativeeffect, or any combination thereof. In some embodiments, the ammonialevel in the subject can be the level of ammonia in the blood (e.g.,plasma) of the subject. In some embodiments, the ammonia-loweringtherapy comprises administering one or more ammonia-lowering agents tothe subject.

Non-limiting examples of ammonia-lowering agents include, or comprise,magnesium phosphate product (MGP), glycerol phenylbutyrate (GPB), sodiumphenylacetate, sodium phenylbutyrate (NaPBA), glutamine, sodiumbenzoate, chlorophyll, L-arabinose, laxatives, antibiotics, ornithine incombination with at least one of phenylacetate and phenylbutyrate, andany combination thereof. The ammonia-lowering agents can be present, forexample, in a pharmaceutical composition, a nutraceutical composition, aprobiotic composition, or any combination thereof. Laxatives aresubstances that can loosen stools and increase bowel movements.Laxatives can be used to lower ammonia levels in gastrointestinal tractof a subject, for example by altering bacterial flora in the subject'sgastrointestinal tract and making few organisms available to produceammonia. Examples of laxatives include, but are not limited to,lactulose.

The ammonia lowering agent can be, or comprises, one or moreantibiotics. For example, the ammonia lowering agent can be administeredby the oral route to allow the antibiotic(s) to act in thegastrointestinal tract. Without being bounded by any particular theory,it is believed that the antibiotic(s) can reduce ammonia-producingbacteria from the intestine to reduce the ammonia level in the subject.Non-limiting examples of the antibiotics include neomycin, vancomycinand rifaximin (Xifaxan).

In some embodiments, different ammonia lowering agents are used incombination to reduce the ammonia level in the subject. For example, oneor more laxatives and one or more antibiotics can be administered to thesubject to reduce ammonia level in the subject.

As another non-limiting example, the ammonia-lower therapy can be, orcomprise, adjusting the composition of gut microbiota in the subject. Insome embodiments, adjusting the composition of gut microbiota of thesubject comprises bacterial transplantation, such as fecaltransplantation. In some embodiments, adjusting the composition of gutmicrobiota in the subject comprises increasing the level of one or morebacterial species lacking or having low urase activity in the gutmicrobiota of the subject. In some embodiments, adjusting thecomposition of gut microbiota in the subject comprises replacing thenative gut microbiota of the subject with a composition having highlevel of one or more bacterial species lacking or having low uraseactivity. In some embodiments, adjusting the composition of gutmicrobiota in the subject comprise administering to the subject acomposition comprising one or more bacterial species lacking or havinglow urase activity. Examples of the bacteria lacking or having lowurease activity include, but are not limited to, Parabacteroides,Lachnospiraceae, Ruminococcaceae, Eubacterium, Mucispirillum,Lactobacillus, and Clostridium. In some embodiments, the bacterialacking or having lower urease activity is Clostridia, Mucispirillumschaedleri, Parabacteroides, Lactobacilli, or any combination thereof.In some embodiments, the ammonia-lowering therapy comprisestransplanting Schaedler flora (ASF), which consists of 8 murine gutcommensal bacterial strains that were assembled in the 1970s andstandardized by the National Cancer Institute in 1978 (Dewhirst et al.,Appl. Environ Microbiol. 1999; 65(8):3287-3292, to the subject. Withoutbeing limited by any particular theory, it is believed that bacterialurease converts host-derived urea to ammonia and carbon dioxide,contributing to hyperammonemia and gut microbiota having no or reducedurease activity can reduce ammonia production and thus ammonia level inthe subject.

In addition, the ammonia-lower therapy can be, or comprise, gene therapyto correct gene defects that contribute to hyperammonenia in thesubject. For example, hyperammonemia can be caused by defects in genesencoding enzymes involved in the urea cycle, including but not limitedto, Ornithine Transcarbamylase (OTC) gene, Carbamyl Phosphate Synthetase(CPS1) gene, Argininosuccinic Acid Synthetase (AAS), ArgininosuccinateLyase (ASL), and Arginase (AG). Hyperammonemia can also be caused bydefects in cystathione beta synthase (CBS) gene and glutamine synthetasegene. The gene therapy can be performed by methods known in the art. Forexample, recombinant viral vectors (e.g., adeno-associated viral vectorsand baculovius vectors) can be used to deliver (e.g., targeted deliveryto liver cells) the missing gene(s) to the subject to reduce the ammonialevel in the subject. See e.g., Torres-Vega et al. Gene Therapy (2015)22, 58-64 (the entire content of which is incorporated herein byreference). In some embodiments, AAV vectors comprising the intact OTCgene are administered into a subject in need thereof to reduce theammonia level in the subject. In some embodiments, AAV vectorscomprising the intact glutamine synthetase gene are administered into asubject in need thereof to reduce the ammonia level in the subject.

Treatment and Prevention of Diseases Associated with HSC Activation

Hepatic stellate cells (HSCs) are liver-specific mesenchymal cells thatplay important roles in liver physiology and fibrogenesis andmaintaining architectural integrity of the liver. HSCs are generallylocated in the space of Disse and maintain close interactions withsinusoidal endothelial cells and hepatic epithelial cells. HSCsorchestrate many important functions in the liver and their dysfunctionis associated with various pathological conditions. HSCs can impact thedifferentiation, proliferation, and morphogenesis of other hepatic celltypes during liver development and regeneration.

In normal liver, HSCs are in a quiescent state. HSCs can change into anactivated state when the liver is damaged. For example, following acuteor chronic liver injury, HSCs undergo phenotypic transformation from“quiescent” (non-proliferating and non-contractile) to “activated”(promitogenic, profibrogenic, and proinflammatory Myofibroblasts-like)cells. Moreover, during the process of activation, HSCs become highlycontractile and have the necessary machinery to contract or relax inresponse to a number of vasoactive substances/stimuli. Activated HSCscan produce a wide array of cytokines and chemokines which may directlyenhance the proliferation of liver progenitor cells and hepatocytes.HSCs are involved in, for example, fibrosis and liver cancerdevelopment. HSC activation can lead to various diseases, conditions andsymptoms, including but not limited to, non-alcoholic fatty liverdisease (NAFLD), fibrotic conditions (for example liver fibrosis), livercancer, and any combination thereof. Non-limiting examples of livercancer include hepatocellular carcinoma (HCC) and hepatoblastoma. Insome embodiments, the methods of treating and/or preventing diseasesassociated with HSC activation comprise identifying a subject sufferingfrom or at the risk of developing a disease associated with HSCactivation. In some embodiments, the disease associated with HSCactivation can be NAFLD, liver fibrosis, liver cancer, or anycombination thereof.

NAFLD refers to a group of conditions where there is accumulation ofexcess fat in the liver of people who drink little or no alcohol. NFALDis a common liver disorder in developed countries. The most common formof NAFLD is a non-serious condition called fatty liver. NAFLD occurswhen fat is deposited (steatosis) in the liver. Although having fat inthe liver is not normal, by itself it probably does not damage theliver. NAFLD is a common cause of fibrosis. NAFLD is sometimes suspectedin an overweight or obese person who is found to have mild elevations intheir liver tests during a routine blood testing or incidentallydetected on radiologic investigations such as abdominal ultrasound or CTscan.

Non-alcoholic steatohepatitis (NASH) is a more serious form of NAFLD. InNASH, fat accumulation is associated with liver cell inflammation anddifferent degrees of scarring. NASH is a potentially serious conditionthat may lead to severe liver scarring and cirrhosis. Without beingbound by any particular theory, it is believed that NASH is associatedwith reduced expression and function of ornithine transcarbamoylase(OTC, also called ornithine carbamoyltransferase) in humans and rodents.For example, in experimental NASH, gene and protein expression of themitochondrial urea cycle enzyme ornithine transcarbamylase (OTC) isreduced significantly, resulting in functional reduction in the in vivocapacity for ureagenesis, which results in hyperammonemia. In patientswith biopsy-proven NASH, plasma ammonia levels are increasedsignificantly more than in patients with simple steatosis. In mammals,the OTC enzyme is part of the urea cycle. In a mammal deficient in OTC,ammonia level will build up, which can cause hyperammonemia andsubsequently neurological problems.

It is disclosed for the first time in the present disclosure thatammonia produces marked morphological and functional changes in humanHSCs and in vivo in bile duct ligated rats (for example, oxidativestress, increased cytokines, expression of activation markers,alterations in the secretion of matrix proteins, and severemorphological disruption). Without being bound by any particular theory,it is believed that hyperammonia can activate HSCs in vivo and in vitro,which may favor the progression of NAFLD (e.g., NASH) and fibrosis. Asdescribed herein, a reduction in ammonia level in a subject can preventthe activation of HSCs in the subject and reduces, for example, diseasesassociated with HSC activation.

Some embodiments described herein provide methods of treating a diseaseassociated with HSC activation in a subject in need by performing on thesubject an ammonia-lowering therapy. Some embodiments described hereinprovide methods of delaying the onset or progression of a diseaseassociated with HSC activation in a subject in need by performing on thesubject an ammonia-lowering therapy. In some embodiments, performing theammonia-lowering therapy comprises administering an ammonia-loweringagent to the subject. In some embodiments, the ammonia-lowering agentis, or comprises, magnesium phosphate product (MGP), glycerolphenylbutyrate (GPB), sodium phenylacetate, sodium phenylbutyrate(NaPBA), glutamine, sodium benzoate, L-arabinose, a laxative, anantibiotic, ornithine in combination with at least one of phenylacetateand phenylbutyrate, or any combination thereof. In some embodiments, themethods comprise co-administering to the subject ornithine incombination with phenylacetate and/or phenylbutyrate. In someembodiments, the disease associated with HSC activation is NAFLD, forexample NASH or steatosis. In some embodiments, the disease associatedwith HSC activation is liver cancer, for example HCC or hepatoblastoma.In some embodiments, the disease associated with HSC activation is afibrotic condition, for example liver fibrosis. In some embodiments, thesubject suffering from liver cancer and/or the fibrotic condition cansuffer from NAFLD as well. In some embodiments, two or moreammonia-lowering agents are co-administered to the subject. In someembodiments, one or more ammonia-lowering agents are co-administeredwith another pharmaceutically active ingredient to the subject. In someembodiments, the composition of gut microbiota in the subject isadjusted to treat a disease associated with HSC activation. In someembodiments, gene therapy is used as the ammonia-lowering therapy totreat a disease associated with HSC activation.

Also disclosed herein are methods of preventing NAFLD by performing anammonia-lowering therapy on a subject in need thereof. In someembodiments, performing the ammonia-lowering therapy compriseadministering an ammonia-lowering agent to the subject. Any of theammonia-lowering agents disclosed herein can be used in the methods,including but not limited to, magnesium phosphate product (MGP),glycerol phenylbutyrate (GPB), sodium phenylacetate, sodiumphenylbutyrate (NaPBA), glutamine, sodium benzoate, L-arabinose,laxatives, antibiotics, ornithine in combination with at least one ofphenylacetate and phenylbutyrate, and any combination thereof. In someembodiments, the methods comprise co-administering to the subjectornithine in combination with phenylacetate and/or phenylbutyrate. Insome embodiments, the composition of gut microbiota in the subject isadjusted to prevent NAFLD. In some embodiments, gene therapy is used asthe ammonia-lowering therapy to prevent NAFLD.

Some embodiments described herein provide methods of treating a fibroticcondition by performing an ammonia-lowering therapy on a subject in needthereof. The ammonia-lowering therapy can comprise, in some embodiments,co-administering to a subject in need thereof an ammonia-lowering agent,such as ornithine in combination with phenylacetate and/orphenylbutyrate. Some such embodiments include therapeutic treatment.Other embodiments include prophylactic treatment. As used herein, a“fibrotic condition” refers to a condition, disease or disorder that ischaracterized by dysregulated proliferation or activity of fibroblastsand/or abnormal accumulation of fibronectin and/or pathologic orexcessive accumulation of collagenous tissue. Typically, any suchdisease, disorder or condition is amenable to treatment byadministration of a compound having anti-fibrotic activity. Fibroticdisorders include, but are not limited to, liver fibrosis (e.g., hepaticfibrosis associated with chronic active hepatitis). Thus, someembodiments include methods of treating liver fibrosis byco-administering to a subject in need thereof ornithine in combinationwith phenylacetate and/or phenylbutyrate. Some embodiments includeidentifying a subject as having or at risk for developing a fibroticcondition (e.g., liver fibrosis) prior to administering the ornithine incombination with phenylacetate and/or phenylbutyrate.

By “co-administration,” it is meant that the two or more agents may befound in the patient's bloodstream at the same time, regardless of whenor how they are actually administered. In some embodiments, the agentsare administered simultaneously. In one such embodiment, administrationin combination is accomplished by combining the agents in a singledosage form. In some embodiments, the agents are administeredsequentially. In some embodiments, the agents are administered throughthe same route, such as orally. In some embodiments, the agents areadministered through different routes, such as one being administeredorally and another being administered i.v.

As described herein, NASH is associated with reduced expression andfunction of the urea cycle enzyme, ornithine transcarbamoylase (OTC)level in humans and rodents, which results in hyperammonemia. Withoutbeing bound by any particular theory, it is believed that ammonia iselevated in NAFLD and is involved in the progression of NAFLD and livercancer. In addition, ammonia-lowering agents (e.g., OP) are useful toreduce blood ammonia level in the subject having hyperammonemia, such asthe hyperammonemia associated with NASH, and thus prevent, limit, orslow down progression of NAFLD, fibrosis progression in NASH, and thedevelopment of liver cancer (e.g., HCC). In some embodiments, theammonia-lowering agent is useful to reduce blood ammonia level, whichtreats and delays the onset or progression of NASH.

Some embodiments include treating a fibrotic condition (e.g., liverfibrosis) by performing an ammonia-lowering therapy on a subject inneed. For example, the ammonia-lowering therapy can compriseadministering to a subject in need an ammonia-lowering agent, forexample ornithine in combination with phenylacetate and/orphenylbutyrate. Some such embodiments include therapeutic treatment.Some embodiments include prophylactic treatment. Some embodimentsinclude identifying a subject as having or at risk for developing thefibrotic condition (e.g., liver fibrosis) prior to administering theammonia-lowering agent.

Some embodiments include methods of treating a liver cancer byperforming an ammonia-lowering therapy on a subject in need. Forexample, the ammonia-lowering therapy can comprise co-administering to asubject in need thereof an ammonia-lowering agent, for example ornithinein combination with phenylacetate and/or phenylbutyrate. Some suchembodiments include therapeutic treatment. Some embodiments includeprophylactic treatment. Some embodiments include identifying a subjectas having or at risk for developing liver cancer prior to administeringthe ammonia-lowering agent. Some embodiments include treating livercancer by administering ornithine in combination with phenylacetateand/or phenylbutyrate, for example ornithine phenylacetate, to thesubject. The liver cancer can be, for example, HCC or hepatoblastoma.

Ammonia level in a subject can be determined by various conventionalmethods. For example, ammonia is routinely measured in plasma from avenous (or arterial) blood sample. It can also be measured in wholeblood, erythrocytes, saliva, sweat, and urine. Ammonia measurements canbe used to diagnose hyperammonemia. Ammonia can be measured by indirector direct methods. For example, the ammonia can be measured by thechange of color of an ammonium indicator, for example the Vitros® (OrthoDiagnostic Ltd.) ammonia measurement which utilizes bromophenol blue. Asanother example, an NH4⁺-selective membrane which is typically based ona mixture of antibiotics nonatin and monoactin can also be used tomeasure ammonia level. In some embodiments, the methods disclosed hereincomprise determining the ammonia level in the subject prior to and/orafter administration of the ammonia-lowering agent. In some embodiments,the ammonia level in the subject is monitored throughout the period inwhich the subject is receiving the treatment by ammonia-lowering agent.Reduction in ammonia levels in vivo can reduce inflammation (NFκB),oxidative stress and αSMA expression, and increase in nitric oxidesynthase (eNOS) activity and function.

Salts

In some embodiments, the ammonia-lowering agents (such as ornithine incombination with phenylacetate and/or phenylbutyrate) are administeredas pharmaceutically acceptable salts. The term “pharmaceuticallyacceptable salt” refers to salts that retain the biologicaleffectiveness and properties of a compound and, which are notbiologically or otherwise undesirable for use in a pharmaceutical. Inmany cases, the ammonia-lowering agents disclosed herein are capable offorming acid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto. Pharmaceutically acceptableacid addition salts can be formed with inorganic acids and organicacids. Inorganic acids from which salts can be derived include, forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, phosphoric acid, and the like. Organic acids from which salts canbe derived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceuticallyacceptable salts can also be formed using inorganic and organic bases.Inorganic bases from which salts can be derived include, for example,bases that contain sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum, and the like;particularly preferred are the ammonium, potassium, sodium, calcium andmagnesium salts. In some embodiments, treatment of the compoundsdisclosed herein with an inorganic base results in loss of a labilehydrogen from the compound to afford the salt form including aninorganic cation such as Li⁺, Na⁺, K⁺, Mg²⁺ and Ca²⁺ and the like.Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. Many such salts are known in the art, as described in WO87/05297, Johnston et al., published Sep. 11, 1987 (incorporated byreference herein in its entirety).

In some embodiments, ornithine is administered as the ornithine HClsalt. In some embodiments, phenylacetate or phenylbutyrate isadministered as their sodium salts. In some embodiments, ornithine andphenylacetate or phenylbutyrate are administered as salts of each other(e.g., ornithine phenylacetate).

Pharmaceutical Compositions and Routes of Administration

The ammonia-lowering agent (such as ornithine in combination withphenylacetate and/or phenylbutyrate) can be formulated foradministration with a pharmaceutically acceptable carrier or diluent.The ammonia-lowering agent can, in some embodiments, be formulated as amedicament with a standard pharmaceutically acceptable carrier(s) and/orexcipient(s) as is routine in the pharmaceutical art. The exact natureof the formulation will depend upon several factors including thedesired route of administration. For example, the ammonia-lowering agent(for example, ornithine and the phenylacetate and/or phenybutyrate) canbe formulated for oral, intravenous, intragastric, intravascular orintraperitoneal administration. Standard pharmaceutical formulationtechniques may be used, such as those disclosed in Remington's TheScience and Practice of Pharmacy, 21st Ed., Lippincott Williams &Wilkins (2005), incorporated herein by reference in its entirety.

The ornithine (e.g., L-ornithine) and phenylacetate or phenylbutyratemay be administered separately or in a single dosage form. In someembodiments, the combination is administered as the ornithinephenylacetate salt or as a solution of the ornithine phenylacetate salt.

Different forms of composition of ornithine in combination with at leastone of phenylacetate (or phenyl acetate salts) and phenylbutyrate havebeen described in U.S. Patent Publication Nos. US2008/0119554 andUS2010/0280119, which are hereby incorporated by reference in theirentireties. In some embodiments, ornithine and phenylacetate is presentand/or administered as ornithine phenyl acetate or physiologicallyacceptable salt thereof. In some embodiments, ornithine is presentand/or administered as a free monomeric amino acid or physiologicallyacceptable salt thereof. In some embodiments, at least one ofphenylacetate and phenylbutyrate is present and/or administered as asodium phenylacetate or sodium phenylbutyrate. In some embodiments, aphysiologically acceptable salt of ornithine and a physiologicallyacceptable salt of at least one of phenylacetate and phenylbutyrate areadministered to the subject.

As disclosed herein, the ornithine and the phenylacetate and/orphenylbutyrate can be formulated for administration in a pharmaceuticalcomposition comprising a physiologically acceptable surface activeagents, carriers, diluents, excipients, smoothing agents, suspensionagents, film forming substances, coating assistants, or a combinationthereof. In some embodiments, the ornithine and the phenylacetate and/orphenylbutyrate are formulated for administration with a pharmaceuticallyacceptable carrier or diluent. The ornithine and the phenylacetateand/or phenylbutyrate can be formulated as a medicament with a standardpharmaceutically acceptable carrier(s) and/or excipient(s) as is routinein the pharmaceutical art. The exact nature of the formulation willdepend upon several factors including the desired route ofadministration. Typically, ornithine and the phenylacetate and/orphenylbutyrate are formulated for oral, intravenous, intragastric,intravascular or intraperitoneal administration. Standard pharmaceuticalformulation techniques may be used, such as those disclosed inRemington's The Science and Practice of Pharmacy, 21st Ed., LippincottWilliams & Wilkins (2005), incorporated herein by reference in itsentirety.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. In addition, various adjuvants such as are commonly usedin the art may be included. Considerations for the inclusion of variouscomponents in pharmaceutical compositions are described, e.g., in Gilmanet al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis ofTherapeutics, 8th Ed., Pergamon Press, which is incorporated herein byreference in its entirety.

Some examples of substances, which can serve aspharmaceutically-acceptable carriers or components thereof, are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose; powdered tragacanth; malt; gelatin; talc; solidlubricants, such as stearic acid and magnesium stearate; calciumsulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil,olive oil, corn oil and oil of theobroma; polyols such as propyleneglycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginicacid; emulsifiers, such as the TWEENS; wetting agents, such sodiumlauryl sulfate; coloring agents; flavoring agents; tableting agents,stabilizers; antioxidants; preservatives; pyrogen-free water; isotonicsaline; and phosphate buffer solutions.

The choice of a pharmaceutically-acceptable carrier to be used inconjunction with the subject compound is basically determined by the waythe compound is to be administered.

The compositions described herein are preferably provided in unit dosageform. As used herein, a “unit dosage form” is a composition containingan amount of a compound that is suitable for administration to ananimal, preferably mammal subject, in a single dose, according to goodmedical practice. The preparation of a single or unit dosage formhowever, does not imply that the dosage form is administered once perday or once per course of therapy. Such dosage forms are contemplated tobe administered once, twice, thrice or more per day and may beadministered as infusion over a period of time (e.g., from about 30minutes to about 2-6 hours), or administered as a continuous infusion,and may be given more than once during a course of therapy, though asingle administration is not specifically excluded. The skilled artisanwill recognize that the formulation does not specifically contemplatethe entire course of therapy and such decisions are left for thoseskilled in the art of treatment rather than formulation.

The compositions useful as described above may be in any of a variety ofsuitable forms for a variety of routes for administration, for example,for oral, nasal, rectal, topical (including transdermal), ocular,intracerebral, intracranial, intrathecal, intra-arterial, intravenous,intramuscular, or other parental routes of administration. The skilledartisan will appreciate that oral and nasal compositions includecompositions that are administered by inhalation, and made usingavailable methodologies. Depending upon the particular route ofadministration desired, a variety of pharmaceutically-acceptablecarriers well-known in the art may be used. Pharmaceutically-acceptablecarriers include, for example, solid or liquid fillers, diluents,hydrotropies, surface-active agents, and encapsulating substances.Optional pharmaceutically-active materials may be included, which do notsubstantially interfere with the inhibitory activity of the compound.The amount of carrier employed in conjunction with the compound issufficient to provide a practical quantity of material foradministration per unit dose of the compound. Techniques andcompositions for making dosage forms useful in the methods describedherein are described in the following references, all incorporated byreference herein: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10(Banker & Rhodes, editors, 2002); Lieberman et al., PharmaceuticalDosage Forms: Tablets (1989); and Ansel, Introduction to PharmaceuticalDosage Forms 8th Edition (2004).

Various oral dosage forms can be used, including such solid forms astablets, capsules, and granules. Tablets can be compressed, tablettriturates, enteric-coated, sugar-coated, film-coated, ormultiple-compressed, containing suitable binders, lubricants, diluents,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents, and melting agents. Liquid oral dosage forms include aqueoussolutions, emulsions, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules, and effervescentpreparations reconstituted from effervescent granules, containingsuitable solvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, melting agents, coloring agents and flavoringagents.

The pharmaceutically-acceptable carriers suitable for the preparation ofunit dosage forms for peroral administration is well-known in the art.Tablets typically comprise conventional pharmaceutically-compatibleadjuvants as inert diluents, such as calcium carbonate, sodiumcarbonate, mannitol, lactose and cellulose; binders such as starch,gelatin and sucrose; disintegrants such as starch, alginic acid andcroscarmelose; lubricants such as magnesium stearate, stearic acid andtalc. Glidants such as silicon dioxide can be used to improve flowcharacteristics of the powder mixture. Coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. The selection of carriercomponents depends on secondary considerations like taste, cost, andshelf stability, which are not critical, and can be readily made by aperson skilled in the art.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically-acceptable carrierssuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include sodium carboxymethyl cellulose, AVICEL RC-591,tragacanth and sodium alginate; typical wetting agents include lecithinand polysorbate 80; and typical preservatives include methyl paraben andsodium benzoate. Peroral liquid compositions may also contain one ormore components such as sweeteners, flavoring agents and colorantsdisclosed above.

Other compositions useful for attaining systemic delivery of the subjectcompounds include sublingual, buccal and nasal dosage forms. Suchcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol; and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose and hydroxypropylmethyl cellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

For topical use, creams, ointments, gels, solutions or suspensions,etc., containing the compound disclosed herein are employed. Topicalformulations may generally be comprised of a pharmaceutical carrier,co-solvent, emulsifier, penetration enhancer, preservative system, andemollient.

For intravenous administration, the compounds and compositions describedherein may be dissolved or dispersed in a pharmaceutically acceptablediluent, such as a saline or dextrose solution. Suitable excipients maybe included to achieve the desired pH, including but not limited toNaOH, sodium carbonate, sodium acetate, HCl, and citric acid. In variousembodiments, the pH of the final composition ranges from 2 to 8, orpreferably from 4 to 7. Antioxidant excipients may include sodiumbisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate,thiourea, and EDTA. Other non-limiting examples of suitable excipientsfound in the final intravenous composition may include sodium orpotassium phosphates, citric acid, tartaric acid, gelatin, andcarbohydrates such as dextrose, mannitol, and dextran. Furtheracceptable excipients are described in Powell, et al., Compendium ofExcipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998,52 238-311 and Nema et al., Excipients and Their Role in ApprovedInjectable Products: Current Usage and Future Directions, PDA J PharmSci and Tech 2011, 65 287-332, both of which are incorporated herein byreference in their entirety. Antimicrobial agents may also be includedto achieve a bacteriostatic or fungistatic solution, including but notlimited to phenylmercuric nitrate, thimerosal, benzethonium chloride,benzalkonium chloride, phenol, cresol, and chlorobutanol.

The compositions for intravenous administration may be provided tocaregivers in the form of one or more solids that are reconstituted witha suitable diluent such as sterile water, saline or dextrose in watershortly prior to administration. In other embodiments, the compositionsare provided in solution ready to administer parenterally. In stillother embodiments, the compositions are provided in a solution that isfurther diluted prior to administration. In embodiments that includeadministering a combination of a compound described herein and anotheragent, the combination may be provided to caregivers as a mixture, orthe caregivers may mix the two agents prior to administration, or thetwo agents may be administered separately.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired effects and thetherapeutic indication. Typically, dosages may be between about 0.1mg/kg and 4000 mg/kg body weight, for example between about 1 mg/kg and1600 mg/kg body weight. Alternatively dosages may be based andcalculated upon the surface area of the patient, as understood by thoseof skill in the art.

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved. The amount of a composition to be administered will,of course, be dependent on many factors including the subject beingtreated, the severity of the affliction, the manner of administration,the judgment of the prescribing physician. The compound or combinationof compounds disclosed herein may be administered orally or viainjection at a dose from 0.1 mg/kg to 4000 mg/kg of the patient's bodyweight per day. The dose range for adult humans is generally from 1 g to100 g/day. Tablets or other forms of presentation provided in discreteunits may conveniently contain an amount of the compound or combinationof compounds disclosed herein which is effective at such dosage or as amultiple of the same, for instance, units containing 1 g to 60 g (forexample, from about 5 g to 20 g, from about 10 g to 50 g, from about 20g to 40 g, or from about 25 g to 35 g). The precise amount of compoundadministered to a patient will be the responsibility of the attendantphysician. However, the dose employed will depend on a number offactors, including the age and sex of the patient, the precise disorderbeing treated, and its severity. Also, the route of administration mayvary depending on the condition and its severity. A typical dose of theammonia-lowering agent (for example, of ornithine, or of phenylacetateor phenylbutyrate) can be from 0.02 g to 1.25 g per kg of body weight,for example from 0.1 g to 0.5 g per kg of body weight, depending on suchparameters. In some embodiments, a dosage of the ammonia-lowering agentcan be from 1 g to 100 g, for example, from 10 g to 80 g, from 15 g to60 g, from 20 g to 40 g, or from 25 g to 35 g. In some embodiments, theornithine and phenylacetate/phenylbutyrate can be administered in aweight ratio from 10:1 to 1:10, for example, from 5:1 to 1:5, from 4:1to 1:4, from 3:1 to 1:3, from 2:1 to 1:2, or about 1:1. A physician willbe able to determine the required dosage of the ammonia-lowering agent(for example, ornithine and of phenylacetate or phenylbutyrate) for anyparticular subject.

The exact formulation, route of administration and dosage for thepharmaceutical compositions of the compound or combination of compoundsdisclosed herein can be chosen by the individual physician in view ofthe patient's condition. (See, e.g., Fingl et al. 1975, in “ThePharmacological Basis of Therapeutics,” which is hereby incorporatedherein by reference, with particular reference to Ch. 1). Typically, thedose range of the composition administered to the patient can be fromabout 0.1 to about 4000 mg/kg of the patient's body weight. The dosagemay be a single one or a series of two or more given in the course ofone or more days, as is needed by the patient. In instances where humandosages for compounds have been established for at least some condition,the present disclosure will use those same dosages, or dosages that arebetween about 0.1% and about 5000%, more preferably between about 25%and about 1000% of the established human dosage. Where no human dosageis established, as will be the case for newly-discovered pharmaceuticalcompounds, a suitable human dosage can be inferred from ED₅₀ or ID₅₀values, or other appropriate values derived from in vitro or in vivostudies, as qualified by toxicity studies and efficacy studies inanimals.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicityor organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made. Incases of administration of a pharmaceutically acceptable salt, dosagesmay be calculated as the free base. In some embodiments, the compositionis administered 1 to 4 times per day. Alternatively the compositions ofthe compound or combination of compounds disclosed herein may beadministered by continuous intravenous infusion, preferably at a dose ofeach active ingredient up to 100 g per day. As will be understood bythose of skill in the art, in certain situations it may be necessary toadminister the compound disclosed herein in amounts that exceed, or evenfar exceed, the above-stated, preferred dosage range in order toeffectively and aggressively treat particularly aggressive diseases orinfections. In some embodiments, the compound or combination ofcompounds disclosed herein will be administered for a period ofcontinuous therapy, for example for a week or more, or for months oryears.

In some embodiments, the dosing regimen of the compound(s) orcombination of compounds disclosed herein is administered for a periodof time, which time period can be, for example, from at least about 1week to at least about 4 weeks, from at least about 4 weeks to at leastabout 8 weeks, from at least about 4 weeks to at least about 12 weeks,from at least about 4 weeks to at least about 16 weeks, or longer. Thedosing regimen of the compound(s) or combination of compounds disclosedherein can be administered three times a day, twice a day, daily, everyother day, three times a week, every other week, three times per month,once monthly, substantially continuously or continuously.

EXAMPLES

Some aspects of the embodiments of the present application are disclosedin further detail in the following examples, which are not in any wayintended to limit the scope of the present disclosure.

Example 1 In Vitro Effect on Primary Human HSCs

Primary human hepatic stellate cells (hHSCs) were cultured. Effects ofan NH₄Cl challenge (0.1-10 mM over 24-72 hrs) on hHSC proliferation(BrdU), metabolic activity (MTS assay), viability (Neutral-Red),ultrastructural changes (TE-M) and gene/protein expression(q-PCR/Western blot) were studied. To test recovery, ammonia treatedcells were replenished with glutamine and in separate experiments,pre-treated with L-methionine-sulfoximine (MSO-GS inhibitor) todetermine the importance of glutamine synthetase (GS).

Hyperammonemia in primary hHSCs induced time dependent decreases inproliferation and metabolic activity, whilst inducing cell swelling anda myo-fibroblast-like phenotype even at 50-100 umol/L.Ultrastructurally, ammonia-treated hHSC had dose-dependent intracellularER enlargement and this was reversible by replenishing the culture withL-glutamine. NH₃ inhibition of hHSC proliferation was dependent on GSactivity as MSO and hyperammonemia induced cell detachment andprevention of recovery suggesting that glutamine is important for hHSCsurvival.

These results suggest that hyperammonemia modifies hHSC's and imparts aswollen myofibroblast phenotype, which is reversible upon ammoniareduction. Accordingly, it is anticipated that therapy that reducesammonia can surprisingly prevent and reverse liver fibrosis.

Example 2 In Vivo Effect in BDL Rats

28-day bile duct ligated (BDL) rats were treated with saline orornithine phenylacetate for 5 days. Portal pressure was measured attermination and tissues were harvested for studies. BDL rats withhyperammonemia had increased hepatic expression of pro-fibrogenichHSC-related genes (α-SMA, PDGFb-R, Myosin IIA/IIB and Coll1), low eNOSactivity and DDAH-1, and high portal pressure, all of which werecorrected by treatment with ornithine phenylacetate.

These results demonstrate that in vivo ammonia lowering with ornithinephenylacetate decreases pro-fibrogenic and activated HSC gene andprotein expression. This data supports the use of ornithinephenylacetate in the treatment (including prevention) of liver fibrosisand liver cancer (stellate cell activation can result in liver cancer).

Example 3 Ammonia Modulates Human HSC Activation

This example shows that ammonia produces deleterious morphological andfunctional effects on HSCs, and ammonia-induced dysfunction of HSCs isreversible using an ammonia-lowering agent OP.

Methods

In this example, primary human HSCs (hHSCs) were isolated and cultured.Proliferation (BrdU), metabolic activity (MTS), morphology (TEM, light-and immunofluorescence microscopy), HSC activation markers, ability tocontract, and changes in oxidative status (ROS) were evaluated toidentify effects of ammonia challenge (50 μM, 100 μM, 300 μM) over 24-72hours. Changes in plasma ammonia levels, markers of HSC activation,portal pressure and hepatic eNOS activity were quantified inhyperammonemic BDL animals, and after OP treatment.

In Vitro Studies in Human HSC

Primary hHSCs were isolated from wedge sections of liver tissue,obtained from patients undergoing surgery in the Royal Free Hospitalafter giving informed consent (EC01.14-RF). Cells were isolatedaccording to Mederacke et al. (Nature Protocols 2015, 10:305-315) withmodifications for human liver as described in Rombouts K, Carloni V.Determination and characterization of tetraspanin-associatedphosphoinositide-4 kinases in primary and neoplastic liver cells. In:Waugh MG, editor. Lipid Signaling Protocols, 2 ed. New York: SpringerScience+Business Media; 2015. p. 203-212). Briefly, 10 g of total humanliver tissue was digested with 0.01% Collagenase, 0.05% Pronase and0.001% DNase I without performing perfusion. The homogenate was filteredthrough a 100 μm cell strainer and the flow-through was centrifuged at50×g for 2 minutes at 4° C. After washing the supernatant, gradientcentrifugation was performed at 1400×g for 17 minutes at 4° C. using an11.5% Optiprep gradient. Finally, the interface was collected andwashed. Purity of hHSCS was established by detection of CD140b(PDGFRbeta), CD29 (Integrin beta 1) and Cytoglobin (CYGB).

The obtained HSCs were cultured in RPMI supplemented with 20% fetalbovine serum (FBS), GLUTAMAX, nonessential amino acids 1×, 1.0 mM sodiumpyruvate, 1× antibiotic-antimycotic (all Life Technologies), referred toas complete HSC medium hereinafter. Experiments described in this studywere performed on hHSCs of at least three independent cell preparationsbetween passage 3 and 8.

Cells were seeded (density 26×10³/cm²) under basic serum-rich conditions(CM complete medium) for 24 hours, followed by serum deprivation foranother 24 hours (SFM). Exogenous glutamine was removed from the culturemedium to avoid uncontrolled generation of ammonia. Specific treatmentwith NH₄Cl treatments were replaced daily for the duration of theexperiment.

Animal Models

All animal experiments were conducted according to the Home Officeguidelines under the UK Animals in Scientific Procedures Act 1986 withapproval of the ethical committee for animal care of University CollegeLondon. This study was performed in male Sprague-Dawley rats (CharlesRiver UK, Margate, UK), weighing 220-250 g.

In one experimental model, rats were administered a highprotein/ammoniagenic diet (AAs) for 5 days. Furthermore, all ratsunderwent BDL to induce cirrhosis or a sham operation as describedpreviously.

Study design. (i) In the first protocol, the prior in vitro observationsof ammonia-induced effects on HSC cell biology were further explored invivo. In this experimental protocol, animals underwent BDL surgery andwere given 4 weeks to develop liver injury. During the 4th week, BDLanimals were randomized into 3 groups: one group contained BDL ratsreceiving an amino acid-rich (AAs) diet in addition to injection ofintraperitoneal (i.p.) saline solution (n=4); a second group receivedthe AAs diet and was treated with an i.p. injection of theammonia-lowering agent ornithine phenylacetate (OP) 0.3 g/kg twice a dayfor 5 days (n=4); the third group consisted of BDL rats receiving salinesolution i.p. (n=4). In addition to the BDL animals, a further group ofsham-operated rats received saline solution (i.p.) (n=4). Animals weresacrificed on the 5th day of treatment.

(ii) In a second protocol, the effect of the ammonia-lowering agent OPon ammonia-induced portal hypertension was investigated. Four weeksafter BDL or sham operation, rats were randomized into three groups:sham-operated rats receiving saline (i.p.) (n=18) twice a day for theexperimental period of 5 days; BDL rats (n=20) were administered i.p.saline twice a day for 5 days; a further group of BDL rats (n=11)received i.p. injection of OP 0.3 g/kg twice a day for 5 days. Betweenweeks 4 and 5, following anesthesia (2% isofluorane), rats from eachgroup underwent assessment of mean arterial pressure via isolation andcannulation of the right carotid artery. In addition, portal pressurewas measured by direct cannulation of the main portal vein. Allmeasurements were transduced to a Powerlab (4SP) linked to Chart v5.0.1software. The mean of three readings taken one minute apart wasrecorded. Liver tissue was harvested and snap-frozen for storage at −80°C. until analyzed.

Statistical Analysis

Results were expressed as mean values±SEM and compared using one-wayanalysis of variance followed by Dunnet's or Tukey's multiple comparisonpost hoc tests, where appropriate. P values<0.05 were consideredsignificant.

In vivo experimental data were analyzed by t tests and Mann-Whitney Utest as appropriate; P<0.05 was considered statistically significant.Results are presented as mean values±SEM using GraphPad Prism software(GraphPad, La Jolla, Calif.)

Results

Ammonia Reduces Cell Proliferation and Metabolism in Human HepaticStellate Cells (hHSCs) In Vitro in a Dose Dependent Manner.

hHSCs treated with different concentrations of ammonia for 72 hoursshowed a significant inhibition in cell proliferation (BrdU assay) andmetabolic activity (MTS assay) (FIG. 1A). Furthermore, long termtreatment of cells with ammonia did not cause cell death in hHSCs asassessed by deploying the Cell Death Detection ELISA (FIG. 1B). Also,these ammonia-induced effects coincided with strong alterations incellular morphology in a dose-dependent manner as observed by lightmicroscopy (FIG. 1C). hHSCs, known as myofibroblast-like cells, as shownin complete medium and under serum starvation changed their morphologydrastically into a spindle-like fibroblast phenotype, with signs ofderegulation of the endo-lysosomal compartment when treated with ammoniaas assessed by Neutral Red, a dye retained by the lysosomes (FIG. 1C).It was found that hHSCs express glutamine synthetase (GS) at the mRNAand protein level. Pretreatment of cells with L-Methionine sulfoximine(MSO, a biochemical inhibitor of GS), followed by exposure to ammoniadid not further inhibit proliferation and metabolic activity incomparison to MSO treatment only.

Ammonia induces alterations in cytoplasmic stress, which coincides withchanges in cellular metabolism/function and actin cytoskeletonarchitecture. The morphological changes observed by light microscopywere further characterized by performing ultrastructural studies.Ammonia caused a dramatic dose-dependent change in the cytosol andmarked presence of translucent vacuoles. Neither mitochondrialalterations nor presence of autophagic structures (characterized bydouble membranes) were observed (FIG. 2A). It was noted that whenammonia-rich medium was removed and cells were replenished with completemedium both cell proliferation and metabolic activity were restored(FIG. 2B), thus supporting that the observed effect of ammonia istransient. Moreover, ammonia-treated hHSCs cultured on collagen gelsshowed a significant ability to contract (FIG. 2C, 2D) when compared tocontrol, and this occurred after 3 hours and was sustained after 24hours of ammonia treatment which coincided with the previously observedmorphological changes (FIG. 2E). Furthermore, long-term treatment withammonia (72 hours) induced a dose-dependent disruption of filamentousactin in the cytoskeleton when TRICT-Phalloidin staining was employed.Re-organization of the F-actin network coincided with the presence oftranslucent vacuoles in a dose dependent manner (FIG. 2F).

Hyperammonemia Induces ROS Production in hHSC.

Prolonged treatment of cells with ammonia for up to 72 hours showed agradual development of ROS as detected by the presence of cytosoliccarboxy-DCF (FIG. 3A). The development of ammonia-induced ROS productionwas further quantitatively measured as described in Mookerjee et al.,(Gastroenterology, 2007, 132:2533-2541) and confirmed that primary hHSCstreated with ammonia produced significant reactive oxygen species (ROS)(FIG. 3B). Next, cells treated with ammonia for different time pointsshowed a strong increase in mRNA expression of Superoxide dismutase 2(SOD2) after 3 hours, which was sustained at 24 hours of ammoniatreatment (FIG. 3C). Moreover, pre-treatment with N-acetyl cysteine(NAC), a known ROS scavenger, showed no impact on the previouslyobserved increase in SOD2 mRNA expression after 3 hours of ammoniatreatment. In contrast, pre-treatment with NAC followed by ammoniatreatment for 24 hours, almost completely abolished ammonia-induced SOD2mRNA expression.

Ammonia Alters the Pro-Fibrogenic/Pro-Inflammatory Profile in hHSCs.

As shown in FIG. 4A, ammonia was shown to significantly increase α-SMAprotein expression. At 300 μM ammonia, vimentin (an importantintermediate filament) synthesis was increased. Both Myosin IIa (playsan important role in HSC contraction) and Myosin IIb (implicated in HSCactivation) were significantly modulated by increasing concentrations ofammonia. A dose-dependent response to ammonia was also observed in P-38MAPK expression. Furthermore, PDGFR-β, important in HSC cellproliferation, showed a significant up-regulation under influence ofammonia, whereas Collagen type I showed a tendency to increase byammonia, albeit these effects were not statistically significant (FIG.4A). Furthermore, ammonia induced a strong and significant up-regulationof MMP2 mRNA expression, whereas mRNA expression of TIMP1 wasdown-regulated (FIG. 4B). Moreover, pro-inflammatory Interleukin-1β mRNAexpression was significantly induced when hHSCs were treated withammonia 300 μM for 72 hours (FIG. 4C), whereas ammonia at 50 μM and 100μM doses significantly up-regulated Interleukin 6 mRNA expression level.By contrast, ammonia did not modify Interleukin 8 mRNA expression in HSC(FIG. 4C). These data show that ammonia-induced ROS formation causesalterations in HSC-related activation markers and pro-inflammatorygenes.

Bile Duct Ligation and Ammonia Treatment Modifies HSC Cell Biology InVivo.

The effect of hyperammonemia on HSC-related signaling pathways in wholeliver tissue was investigated. Ammonia concentrations are significantlyelevated in BDL rat plasma compared to sham-operated rats (149.3μmol/L±51.1 vs. 107.4 μmol/L±23.2, P<0.05). Plasma ammonia levelsfurther increased when animals were fed an amino acid-rich (AAs) diet incombination with BDL surgery (199.1 μmol/L±43.6 vs. 149.3 μmol/L±51.1,P<0.05) (FIG. 5A). More importantly, plasma ammonia levels decreasedsignificantly when BDL-AAs-fed animals were treated with OP (123.9μmol/L±16.1 vs.199.1 μmol/L μM±43.6, P<0.001) (FIG. 5A).

OP treatment was found to result in a marked decrease in proteinexpression of HSC-related activation markers (FIG. 5B). Morespecifically, BDL in combination with hyperammonaemia (AAs diet) showeda significant increase in Myosin IIb, Collagen type I, and PDGF-Rβprotein expression in comparison to BDL. In contrast, treatment with OPabrogated the strong effect of hyperammonemia on BDL rat livers inrelation to all HSC-related activation markers tested (FIG. 5B).

The example shows that pathophysiological ammonia concentrations causedsignificant and reversible changes in cell proliferation, metabolicactivity and activation markers of hHSCs in vitro. Ammonia also inducedsignificant alterations in cellular morphology, characterized bycytoplasmic vacuolization, ROS production, hHSC contraction and changesin pro-inflammatory gene expression together with HSC-related activationmarkers such as α-SMA, myosin IIa, IIb, and PDGF-Rβ. Treatment with anammonia-reducing agent OP significantly reduced plasma ammonia (BDL199.1 μmol/L±43.65 vs. BDL+OP 149.27 μmol/L±51.1, P<0.05), which wasassociated with increased eNOS activity and abrogation of HSC activationmarkers.

Example 4 OTC Gene Expression and Hepatic Urea Nitrogen Handling areReduced in NAFLD Animals and Recovers with Dietary Modulation andReducing Bacterial Translocation

This example shows that gene and protein expression of ornithinetranscarbamylase (OTC) are altered in animal models of NASH and thealteration is reversible with recovery of animals by restoring the dietand by reducing bacterial translocation. This example also shows thatgene expression of OTC is altered in NAFLD patients.

Two animal models of NASH were studied: a) Wistar rats were fed ahigh-fat, high-cholesterol diet (HFHC) for 10 months and then recoveredfor 2 months on standard diet, and b) Mice were fed a methionine-cholinedeficient diet (MCD) for 4 weeks and treated with Yaq-001 (Yaqrit Ltd.),a nanoporous carbon which has been shown to reduce bacterialtranslocation. In addition, liver biopsies from 16 NAFLD human patientswere obtained during bariatric surgery and the OTC gene expression inthose liver biopsies was measured.

In both of the NASH animal models, gene and protein expression of OTCwas reduced significantly and the reduction was restored by dietarymodulation or reduction in bacterial translation. For example, in theHFHC rats, reversal of NASH by changing the diet to normal chow restoredOTC gene expression (0.53 (CI 0.41-0.68) vs. 0.32 (CI 0.28-0.37),P<0.05; controls 1.00 (CI 0.85-1.17)) and OTC protein expression(5.33±0.21 vs. 3.06±0.20, P<0.01; controls 7.41±0.68) (FIG. 6). In theMCD mice, reduction in bacterial translocation prevented development ofNASH and restored OTC gene expression (0.89 (CI 0.13-0.16) vs. 0.35 (CI0.08-0.09), P<0.01; controls 1.00 (CI 0.12-0.17)) suggesting thatinflammation in NASH contributes to OTC gene expression (FIG. 7).

In the NAFLD patients, those with NASH and fibrosis had significantlylower OTC gene expression than patients with steatosis alone (0.82±0.37vs. 1.15±0.24, P=0.05) (FIG. 8).

As shown in this example, experimental and human NASH resulted in areduction in gene expression of the urea cycle enzyme OTC impairingnitrogen homeostasis. The changes were reversible in the animal modelsof NASH with dietary intervention and also by reducing bacterialtranslocation. The results shown herein indicate a link between NASH,reduction in gene expression and function of OTC and bacterialtranslocation. Moreover, ammonia produces morphological changes andactivation of HSCs, and OTC reduction can result in hyperammonemia andprogression of liver injury and fibrosis. This example supportstargeting ammonia and bacterial translocation as treatments for NASH.

Example 5 Hyperammonemia Leads to Disease Progression and Administrationof Ammonia-Reducing Agent Reduces Progression of NASH and Fibrosis

Two Animal Models are studies in this example: (i) Sprague Dawley ratsare subdivided and fed either a diet enriched in High Fat and HighCholesterol (HFHC diet) or a standard diet without high fat andcholesterol content (Standard diet) for up to 16 weeks; and (ii) Ratsare fed a High Fat diet supplemented with Fructose (HFD+F diet) for upto 16 weeks.

Interventions study: (i) Prevention therapy—OP are given simultaneouslywith the fat supplemented diets to the rat;

(ii) Interventional therapy starts 8 weeks after diet-induced NASH tomimic a therapeutic intervention in established NASH rat model.Subgroups are administered OP (0.3 g/kg twice a day, orally) or placebo.In total, 6 experimental groups are investigated: 1) Standarddiet+saline; 2) Standard diet+OP (week 1-16); 3) Standard diet+OP (week8-16); 4) HFHC diet+saline; 5) HFHC diet+OP (week 1-16)—preventivetherapy; and 6) HFHC diet+OP (week 8-16)—interventional therapy.

(iii) Exaggerated hyperammonemia: One additional rat group that receivesan amino acid-rich (AAs) diet to induce hyperammonemia serves as apositive control.

All animals are sacrificed and tissues are collected to investigate themechanisms that link OTC dysfunction with NASH development and thepharmacological modulation of hyperammonemia.

Primary end point of these experiments is to ascertain the severity ofNASH and fibrosis in the various groups studied. For NASH and fibrosisscoring, histological study is performed and the NASH CRN scoring systemis used by an experienced hepato-pathologist blinded to the type oftreatment received by the animals (TVL; APD). Commonly describedvariables in NASH are analyzed on Hematoxylin-eosin stained sections: 1)macro/micro vesicular steatosis, 2) lobular inflammation, 3)hepatocellular ballooning, and 4) apoptotic bodies. Fibrosis/collagenaccumulation is assessed using Sirius Red stained sections. In addition,oil red O staining is performed to investigate changes in lipidaccumulation and Filipin staining to observe changes in cholesterol.

For secondary end-points: 1) blood samples: analysis of plasmabiochemistry (serum ALT, AST, urea, ammonia, albumin, cholesterol-LDL,cholesterol-HDL cholesterol and triglycerides) are performed using CobasIntegra 400 multi analyzer with appropriate kits (Roche Diagnostics,Burgess Hill, West Sussex, UK). 2) Measurement of OTC enzyme activityand assessment of OTC (and other urea cycle related enzymes) isperformed using qPCR and Western blot analysis. Changes in OTClocalization/zonation are assessed using immunohistochemistry. 3)Pro-fibrogenic, activation-related HSC markers are detected. 4)Pro-inflammatory cytokines/chemokines and macrophage markers aredetected. And 5) Apoptosis-related markers are detected.

For power calculations and statistical analysis, experiments areundertaken to demonstrate a significant difference between the differentconditions under investigation at a p value of <0.05 with 80% power(using ANOVA with selected post-group comparisons). Previous studiesindicate n=12 animals in each group to be sufficient to demonstrate asignificant change.

It is expected that (i) animal models of NASH simulating the calorie andfat dense Western diet have hyperammonemia and reduced OTC expressionand function; and (2) treatment with an ammonia-reducing agent (forexample OP) reduces biochemical, inflammatory and histological indicesof liver injury and reverses OTC dysfunction and hyperammonemia.

Example 6 Ammonia-Reducing Agent Treats Liver Cancer

In this example, a rat model of fibrosis/HCC is used to determinewhether an ammonia-reducing agent OP can reduce the risk of HCCdevelopment.

Animals are studied up to at 16 weeks and examined for the developmentof HCC. Animals (6-8/group) are treated withdiethylnitrosamine(DEN)/nitrosomorpholine (NMOR) to induce fibrosis/HCCas previously described (Mohamed et al., Liver International 2015,35(3):1063-1076). The six animal groups for study are listed in Table 1.

TABLE 1 Animal groups 1. Sham + saline 2. Sham + OP (week 1-14) -prevention 3. Sham + OP (week 7-14) - treatment 4. DEN + saline 5. DEN +OP (week 1-14) - prevention 6. DEN + OP (week 7-14) - treatment

Example 7 Reduction in Ammonia Level Reduces Progression of NAFLD

NAFLD is induced in rats by feeding male rats with a liquid high-fatdiet (HFD) (71% of kcal fat) for up to 16 weeks. Obese Zucker rats,which is one of the most commonly used models of NAFLD in rats, areprovided. Ornithine in combination with at least one of phenylacetateand phenylbutyrate, for example OP, are administered to the HFD rats andthe obese Zucker rats. It is expected that the administration ofornithine in combination with at least one of phenylacetate andphenylbutyrate, which reduces ammonia concentration in the HFD rats andthe obese Zucker rats, is effective in reducing progression of NAFLD indiet-induced NAFLD rat models as well as in genetic rat model of NAFLD.

Example 8 Hyperammonemia Worsens Progression of NAFLD and Fibrosis

NAFLD is induced in rats by feeding male rats with a liquid high-fatdiet (HFD) (71% of kcal fat) for three weeks. Obese Zucker rats, whichis one of the most commonly used models of NAFLD in rats, are provided.Spontaneous hyperammonemia is engineered in both diet-induced andgenetic NAFLD rat models by making the rats deficient in ornithinetranscarbamoylase (OTC deficiency). OTC deficient rats are made bymutating or deleting the OTC gene in the rats. Induced hyperammonemia isengineered in both diet-induced and genetic NAFLD rat models by feedinghigh-protein diet to the rats. It is expected that both of thespontaneous and induced hyperammonemia worsen the progression of NAFLDand fibrosis.

Example 9 Weight Reduction Reduces Progression of NAFLD

NAFLD is induced in rats by feeding male rats with a liquid high-fatdiet (HFD) (71% of kcal fat) for three weeks. Obese Zucker rats, whichis one of the most commonly used models of NAFLD in rats, are provided.Weight reduction surgery is performed on the HFD rats and the obese. Itis expected that the weight reduction surgery reduces progression ofNAFLD in diet-induced NAFLD rat models as well as in genetic rat modelof NAFLD. It is also expected that the weight reduction improves hepaticnitrogen handling, OTC gene/protein expression and function in the NAFLDrat models.

Although the present disclosure has been described with reference toembodiments and examples, it should be understood that numerous andvarious modifications can be made without departing from the spirit ofthe present disclosure. Accordingly, the present disclosure is limitedonly by the following claims.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited herein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differ from or contradictthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls.

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited herein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differ from or contradictthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls

1.-32. (canceled)
 33. A method of treating or delaying the onset orprogression of a disease associated with hepatic stellate cell (HSC)activation, comprising administering one or more ammonia-lowering agentsto a subject in need thereof, wherein the ammonia-lowering agent isselected from the group consisting of sodium phenylacetate, sodiumphenylbutyrate (NaPBA), sodium benzoate, a laxative, an antibiotic,ornithine in combination with at least one of phenylacetate andphenylbutyrate, and any combinations thereof.
 34. The method of claim33, wherein the disease associated with HSC activation is non-alcoholicfatty liver disease (NAFLD).
 35. The method of claim 34, wherein theNAFLD is non-alcoholic steatohepatitis (NASH).
 36. The method of claim34, wherein the NAFLD is steatosis.
 37. The method of claim 33, whereinthe disease associated with HSC activation is liver cancer.
 38. Themethod of claim 33, wherein the disease associated with HSC activationis a fibrotic condition.
 39. The method of claim 38, wherein thefibrotic condition is liver fibrosis.
 40. The method of claim 33,wherein the ammonia-lowering agent is ornithine phenylacetate.
 41. Themethod of claim 40, wherein ornithine phenylacetate is administered incombination with a laxative.
 42. The method of claim 33, wherein theadministration is oral, intravenous, intraperitoneal, intragastric, orintravascular administration.
 43. The method of claim 42, wherein theadministration is intravenous or oral administration.
 44. A method ofpreventing non-alcoholic fatty liver disease (NAFLD), comprisingadministering one or more ammonia-lowering agents to a subject in needthereof, wherein the ammonia-lowering agent is selected from the groupconsisting of sodium phenylacetate, sodium phenylbutyrate (NaPBA),sodium benzoate, a laxative, an antibiotic, ornithine in combinationwith at least one of phenylacetate and phenylbutyrate, and anycombinations thereof.
 45. The method of claim 44, wherein the NAFLD isnon-alcoholic steatohepatitis (NASH) or steatosis.
 46. The method ofclaim 44, wherein the ammonia-lowering agent is ornithine in combinationwith phenylacetate.
 47. The method of claim 46, wherein separatepharmaceutically acceptable salts of the ornithine and phenylacetate areadministered to the subject.
 48. The method of claim 47, whereinphenylacetate is administered as sodium phenylacetate.
 49. The method ofclaim 47, wherein the ornithine is administered as a free monomericamino acid or physiologically acceptable salt thereof.
 50. The method ofclaim 46, wherein the ornithine in combination with phenylacetate isadministered as ornithine phenylacetate.
 51. The method of claim 44,wherein the administration is oral, intravenous, intraperitoneal,intragastric, or intravascular administration.
 52. The method of claim51, wherein the administration is intravenous or oral administration.